1. Field of the Invention
The present invention relates to a method and kit for determining the presence and quantity of nuclease activity in a sample having an unknown nuclease activity.
2. Background of the Art
The contamination of laboratory equipment and solutions by nucleases is highly problematic in experiments involving nucleic acids, because these contaminating enzymes can break down the nucleic acids essential to the experiment. To avoid this problem, nuclease-free chemicals must be purchased from vendors, and equipment and solutions must be autoclaved or treated with DEPC before and during experimentation.
Contamination is a particular problem for large manufacturing facilities producing molecular biology-grade chemicals, because of quality control protocols. Further, monitoring for contamination by nucleases is needed during the purification and characterization of nucleases.
Assaying for contamination by nucleases is not now routinely carried out because the presently available methods for assaying are too time-consuming, or require expertise or materials not readily available. Of the several heretofore available methods, the most commonly used is monitoring the degradation of DNA and RNA by measuring the variation in optical density at 260 nanometers. This method, however, requires expensive quartz glass cuvettes, and is particularly time-consuming when analyzing a large number of samples. (Blackburn, et al., Journal of Biological Chemistry, Vol. 252, No. 16, (1977)).
Nuclease activity can also be determined by measuring the radioactive decay of .sup.32 P-labeled DNA or RNA. This technique requires multiple steps to separate intact DNA and RNA from digested nucleic acids, and entails the handling of radioactive materials.
Still another method of measuring nuclease activity is to electrophoretically separate digested DNA and RNA from undigested DNA and RNA, and stain the undigested DNA and RNA bands with ethidium bromide. (March and Gonzalez, Nucleic Acids Research, Vol. 18, No. 11, (1990)). This method takes considerable amounts of time and, thus, is not suitable for detection of nuclease activity in a large number of samples.
DNase activity has also been determined using an antibody capture bioassay. In this method, DNase in test samples is trapped in anti-DNase polyclonal antibody-coated microtiter plates. (Gibson, et al., Journal of Immunological Methods 155, 249 (1992)). The difficulty with this technique is that it requires antibodies with specificity against the particular nuclease of interest, and these antibodies have limited availability.
Thus, there remains a need for a method to determine the presence and quantity of nuclease activity in a sample having an unknown nuclease activity that is both cost-effective and capable of being performed in a reasonable period of time.
There are a variety of commercially-available, fluorescing, nucleic acid dyes. These include ethidium bromide, in addition to newer dyes made from the general groups of benzoxazolium-4-pyridinium, benzothiazolium-4-pyridinium, benzoxazolium-4-quinolinium, Yoyo-1, Toto-1, Toto-3.
The effects of ethidium bromide on nuclease activity have been documented in a number of previous experiments. In one study, ethidium bromide intercalation with DNA in chromatin increased the digestion rate by micrococcal nuclease. However, ethidium bromide intercalation with free DNA inhibits the digestion rate by micrococcal nuclease. In the former case, the micrococcal nuclease recognizes sites different from that of non-intercalated DNA in chromatin. (Jerzmanowski, et al., Biochimica et Biophysica Acta, 521:493-501, (1978)). Similarly, pre-treatment of DNA with ethidium bromide caused an alteration in the recognition sites by Exonuclease III on DNA which was combined with the anti-tumor drug cis-diamminedi-chloroplatinum (II). (Tullius & Littard, Proceedings of the National Academy of Science U.S.A., Vol. 72, pp. 3489-3492 (1982)).
In another experiment, ethidium bromide intercalation of DNA was found to decrease the activity of S.sub.1 nuclease. (Alvi, Rizvi, and Hadi, Chemical Biological Interactions, Vol. 53, pp. 219-231 (1985)). Similarly, ethidium bromide intercalation of mitochondrial and plasmid pBR322 DNA inhibited the activity of various restriction endonucleases, but the inhibition varied depending on the restriction site. (Soslau and Pirollo, Biochemical and Biophysical Research Communications, Vol. 115, No. 2 (1983)). An endonuclease purified from chicken liver was found to have increased activity on heat-denatured DNA and on native DNA that was pre-treated with intercalating agents, including ethidium bromide. (Rizvi and Hadi, Indian Journal of Biochemistry and Biophysics, Vol. 19, pp. 394-398 (1982)).
As can be appreciated frown the above experiments, the effects of intercalating agents on nuclease activity, as represented by ethidium bromide, depends on the state of the DNA and the nuclease. The effects of the newer nucleic acid dyes on nuclease activity upon any of the various nucleic acid substrates was, heretofore, unknown.